(1) Field of the Invention
The present invention relates to host cells that include one or more nucleic acid molecules encoding a Ca2+ ATPase, endoplasmic reticulum lectin chaperones, e.g., calreticulin (CRT) or calnexin (CRX), and/or ERp57 protein and their use for producing recombinant glycoproteins that have reduced O-glycosylation.
(2) Description of Related Art
Glycoproteins mediate many essential functions in humans and other mammals, including catalysis, signaling, cell-cell communication, and molecular recognition and association. Glycoproteins make up the majority of non-cytosolic proteins in eukaryotic organisms (Lis and Sharon, Eur. J. Biochem. 218: 1-27 (1993)). Many glycoproteins have been exploited for therapeutic purposes, and during the last two decades, recombinant versions of naturally-occurring glycoproteins have been a major part of the biotechnology industry. Examples of recombinant glycosylated proteins used as therapeutics include erythropoietin (EPO), therapeutic monoclonal antibodies (mAbs), tissue plasminogen activator (tPA), interferon-β (IFN-β), granulocyte-macrophage colony stimulating factor (GM-CSF)5 and human chorionic gonadotrophin (hCH) (Gumming et al., Glycobiology 1:115-130 (1991)). Variations in glycosylation patterns of recombinantly produced glycoproteins have recently been the topic of much attention in the scientific community as recombinant proteins produced as potential prophylactics and therapeutics approach the clinic.
In general, the glycosylation structures of glycoprotein oligosaccharides will vary depending upon the host species of the cells used to produce them. Therapeutic proteins produced in non-human host cells are likely to contain non-human glycosylation which may elicit an immunogenic response in humans—e.g. hypermannosylation in yeast (Ballou, Methods Enzymol. 185:440-470 (1990); α(1,3)-fucose and β(1,2)-xylose in plants, (Cabanes-Macheteau et al, Glycobiology 9: 365-372 (1999)); N-glycolylneuraminic acid in Chinese hamster ovary cells (Noguchi et al., J. Biochem. 117: 5-62 (1995); and, Galα-1,3Gal glycosylation in mice (Borrebaeck et al., Immunol. Today, 14: 477-479 (1993).
Because the oligosaccharide structures of glycoproteins produced by non-human mammalian cells tend to be more closely related to those of human glycoproteins, most commercial glycoproteins are produced in mammalian cells. However, mammalian cells have several important disadvantages as host cells for protein production. Besides being costly, processes for producing proteins in mammalian cells produce heterogeneous populations of glycoforms, have low volumetric titers, and require both ongoing viral containment and significant time to generate stable cell lines. Until about 2000, lower eukaryote host cells suitable for producing recombinant glycoproteins with human-like N-glycosylation patterns had not been possible. Since then, Gerngross in U.S. Pat. No. 7,029,872 disclosed methods for making recombinant lower eukaryote host cells that are capable of making glycoproteins that have human-like N-glycosylation patterns. Thus, there is now considerable interest in using lower eukaryote host cells to produce recombinant glycoproteins.
While the pathway for N-linked glycosylation has been the subject of much analysis, the process and function of O-linked glycosylation is not as well understood. It is known that in contrast to N-linked glycosylation, O-glycosylation is a posttranslational event, which occurs in the cis-Golgi (Varki, Glycobiol., 3: 97-130 (1993)). While a consensus acceptor sequence for O-linked glycosylation like that for N-linked glycosylation does not appear to exist, a comparison of amino acid sequences around a large number of O-linked glycosylation sites of several glycoproteins show an increased frequency of proline residues at positions −1 and +3 relative to the glycosylated residues and a marked increase of serine, threonine, and alanine residues (Wilson et al., Biochem. J., 275: 529-534 (1991)). Stretches of serine and threonine residues in glycoproteins, may also be potential sites for O-glycosylation. It has been shown that yeast-derived recombinant proteins often bear additional unnatural O-glycans compared to their natural counterpart (Van den Steen, et al., Crit. Reviews in Biochem. and Mole. Biol. 33: 151-208, (1998)). These unnatural O-glycans can result in proteins that have unwanted immunogenicity or aberrant activity. Thus, there is a need to develop methods for producing proteins in yeast and other lower eukaryotes that have reduced or no O-glycosylation.
Tanner et al. in U.S. Pat. No. 5,714,377 describes the PMT1 and PMT2 genes of Saccharomyces cerevisiae and a method for making recombinant proteins having reduced O-linked glycosylation that uses fungal cells in which one or more of PMT genes have been genetically modified so that recombinant proteins are produced, which have reduced O-linked glycosylation.
Ng et al. in U.S. Published Patent Application No. 20020068325 discloses inhibition of O-glycosylation through the use of antisense or cosuppression or through the engineering of yeast host strains that have loss of function mutations in genes associated with O-linked glycosylation, in particular, one or more of the PMT genes.
Clausen in U.S. Published Patent Application No. 20030186850 discloses the use of GalNAc-beta-benzyl to selectively inhibit lectins of polypeptide GalNAc-transferases and not serve as substrates for other glycosyltransferases involved in O-glycan biosyntheses, thus inhibiting O-glycosylation.
Orchard et al. in U.S. Pat. No. 7,105,554 describes benzylidene thiazolidinediones and their use as antimycotic agents, e.g., antifungal agents which Bobrowicz et al. in WO2007061631 show can be used in a way which is not lethal to the host cells for production of recombinant proteins with reduced O-linked glycosylation.
Konrad et al. in U.S. Published Patent Application No. 20020128235 disclose a method for treating or preventing diabetes mellitus by pharmacologically inhibiting O-linked protein glycosylation in a tissue or cell.
Kojima et al. in U.S. Pat. No. 5,268,364 disclose therapeutic compositions for inhibition of O-glycosylation using compounds such as benzyle-α-N-acetylgalactosamine, which inhibits extension of O-glycosylation leading to accumulation of O-α-GalNAc, to block expression of SLex or SLea by leukocytes or tumor cells and thereby inhibit adhesion of these cells to endothelial cells and platelets.
Boime et al. in U.S. Pat. No. 6,103,501 disclose variants of hormones in which O-linked glycosylation was altered by modifying the amino acid sequence at the site of glycosylation.
However, even in light of the above attempts to produce recombinant host cells that produce proteins that have reduced or no O-glycosylation, there still remains a need for host cells that are capable of producing recombinant proteins that have reduced O-glycosylation.